The goal of the proposed work is to test the hypothesis that the plasma membrane and intracellular organelle membranes possess a multicomponent membrane skeleton that is crucial to production and maintenance of normal bone tissue. The first aim is to establish the role that spectrin and ankyrin play in the attachment/adhesion mechanism of osteoclasts during resorptive and non-resorptive periods. This objective is based on the immunolocalization of ankyrin to the clear zone of osteoclasts in situ and to a structure associated with the cell-substrate interface in osteoclasts in culture. The second aim, to establish the distribution and interaction of ankyrin in osteoclast organelles and vesicles, is based on the finding of multiple ankyrin isoforms in osteoclast lysates and immunocytochemical localization to organelle and vesicle membranes. Osteoclasts will be isolated from 20-day chick embryos and developed from a murine marrow culture system. The cells will be treated with agents known to alter resorptive behavior or interfere with cadherin function prior to immunolocalization and immunoprecipitation studies. The third aim is to establish the role of ankyrin in bone cells using the murine ankyrin-1 deficient mutant, nb/nb, which has significant low bone density when compared to heterozygous littermate controls. Osteoclast and osteblast cultures will be established using the mutant mice and the normal littermate control animals. Initial studies of bone cell activity parameters include tartrate-resistant acid phosphatase staining and quantitative assessment of resorption pit formation by osteoclasts and alkaline phosphatase staining and production of osteocalcin and type I collagen by osteoblasts. Aberrations in skeletal physiology are reflected in congenital defects apparent at birth and in later onset conditions such as osteoporosis. The proposed studies will generate basic information about osteoclast and osteoblast physiology that will have long-term value in the design of effective therapeutic strategies aimed at modulating bone cell activity.